식품의약품안전청 _KJYDGP001 2007. 11. 13

1. DNA Methylation by EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH)

1-2. The Sample list

1-1. The Method of Methylation

Control DNA_F1, Treated DNA_F1 두 개의 샘플에 대해서 EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH) 을 사용하여 methylation 시킨 뒤 PCR 실험을 수행하였고 ,Negative control 로 methylation 처리를 하지 않은 원상태의 Control DNA_F1, Treated DNA_F1 또한 PCR 실험을 수행하였다

　 Sample Name

1 Control DNA_F1-Mehtylated

2 Treated DNA_F1-Mehtylated

3 Control DNA_F1-unmehtylated

4 Treated DNA_F1-unmehtylated

식품의약품안전청 _KJYDGP001 2007. 11. 13

1-3. Primer list

1. DNA Methylation by EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH)

Primer PCR product size(bp)

1 Cdkn1c-F1-left/right 4282 Cdkn1c-F2-left/right 4293 Mrps18b_1-F1-left/right 2204 Mrps18b_2-F1-left/right 4885 Mrps18b_2-F2-left/right 4116 Mrps18b_3-F1-left/right 6007 Mrps18b_3-F2-left/right 4278 Tmem56_1-F1-left/right 4539 Vasp_1-F1-left/right 30610 Vasp_1-F4-left/right 41011 Vasp_2-F1-left/right 29812 Pctk1-F1-left/right 71913 Pctk1-F5-left/right 77614 Auts2-F1-left/right 51615 Auts2-F2-left/right 86016 Spata3-F1-left/right 32217 Piwil2-F1-left/right 394

2007. 11. 13

2. The Result of Secondary PCR

2-1. PCR reaction combination

식품의약품안전청 _KJYDGP001

2-2. PCR Condition

Temp. Time CyclesFinal

Reaction vol.

95 ℃ 5 min 1

30ul

Ex-Taq

94 ℃ 30 sec

3065 ℃ 30 sec

72 ℃ 1 min

72℃ 5 min 1

4℃ Storage 1

PCR 수행 시 Control DNA _F1-Methylated, Treated DNA_F1-Methylated, Control DNA_F1-unmethylated,Treated DNA_F1-unmethylated 4 개의 DNA 샘플과 17 개의 primer set 을 가지고 primary PCR 수행 후 여기서얻은 PCR product DNA 를 template 로 하여 secondary PCR 을 수행하여 결과를 얻었다

Control DNA Treated DNA Control DNA Treated DNAMrps18b_1-F1-L/R 1 2 3 4Mrps18b_2-F1-L/R 5 6 7 8Mrps18b_2-F2-L/R 9 10 11 12Mrps18b_3-F1-L/R 13 14 15 16Mrps18b_3-F2-L/R 17 18 19 20Tmem56_1-F1-L/R 21 22 23 24

Vasp_1-F1-L/R 25 26 27 28Vasp_1-F4-L/R 29 30 31 32Vasp_2-F1-L/R 33 34 35 36

Pctk1-F1-L/R 37 38 39 40Pctk1-F5-L/R 41 42 43 44Auts2-F1-L/R 45 46 47 48Auts2-F2-L/R 49 50 51 52

Spata3-F1-L/R 53 54 55 56Piwil2-F1-L/R 57 58 59 60

Cdkn1c-F1-L/R 61 62 63 64Cdkn1c-F2-L/R 65 66 67 68

Methylated Unmethylated

1

2007. 11. 13

2-3. PCR Result

2% 0..5X TAE gel

식품의약품안전청 _KJYDGP001

17

41

20

25

53 54

2. The Result of Secondary PCR

2007. 11. 13

2. The Result of PCR

식품의약품안전청 _KJYDGP001

41

1753 54

1kb Ladder

2-4. PCR Result

400bp300bp

800bp

2% 0..5X TAE gel

2% 0..5X TAE gel 2% 0..5X TAE gel

Control DNA Treated DNAMrps18b_3-F2-L/R 17 427

Pctk1-F5-L/R 41 776Spata3-F1-L/R 53 54 322

Methylated PCR productsize (bp)